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Circulation Research. 1999;85:e59-e69

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(Circulation Research. 1999;85:e59.)
© 1999 American Heart Association, Inc.

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Regulation of Cardiac Myocyte Protein Turnover and Myofibrillar Structure In Vitro by Specific Directions of Stretch

D. G. Simpson, M. Majeski, T. K. Borg, L. Terracio

From the Department of Anatomy (D.G.S.), Medical College of Virginia, Virginia Commonwealth University, Richmond, Va; Department of Developmental Biology and Anatomy (M.M., T.K.B., L.T.), University of South Carolina School of Medicine, Columbia, SC.

Correspondence to David G. Simpson, PhD, Virginia Commonwealth School of Medicine, Department of Anatomy, Richmond, VA 23298.


*    Abstract
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 *Abstract
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Abstract—We have examined how different degrees (0.5%, 1.0%, 2.5%, 5.0%, and 10.0%) and directions of stretch regulate the turnover and accumulation of contractile proteins in cultured neonatal cardiac myocytes (NCMs). In pulse-chase experiments, stellate-shaped NCMs with random arrays of myofibrils (MFs) exhibited a threshold response to stretch. With respect to unstretched controls, the turnover of the contractile protein pool was suppressed 50% to 100% in stellate NCMs stretched 1.0% to 5.0% and was unaltered in stellate NCMs stretched 0.5% or 10.0%. The posttranslational metabolism of myosin heavy chain (MHC) and actin was regulated in parallel with the total contractile protein pool. The turnover of the cytoplasmic protein pool remained unchanged in response to stretch. NCMs plated onto an aligned matrix of type I collagen expressed an elongated, rod-like cell shape. The MFs of these cells were distributed in parallel with one another along a single unique axis. The tissue-like pattern of organization of these cultures made it possible to assay how specific directions of stretch affected cardiac protein turnover and MF organization. In pulse-chase experiments, stretch in parallel with the MFs did not alter the turnover of the total contractile protein pool, the cytoplasmic protein pool, MHC, or actin. The total cellular concentration of MHC and actin remained constant, and MF alignment was not overtly affected. In contrast, even modest degrees of stretch across the short axis of the MFs suppressed total contractile protein turnover, the turnover of MHC and actin, and promoted the accumulation of these MF subunits. The parallel alignment of MFs deteriorated in myocytes stretched greater than 5%. The characteristic response of aligned myocytes to stretch was not affected by the contractile state of the cells. Isoproterenol (ISO) treatment in concert with stretch in parallel with the MFs modestly accelerated contractile protein turnover. Conversely, contractile protein turnover was suppressed in cells treated with ISO and stretched across the short axis of the MFs. Contractile arrest with nifedipine (NIFED) accelerated total myofibrillar protein turnover. Stretch across the short axis, but not in parallel with the MFs, suppressed protein turnover in cells treated with NIFED. The turnover of the cytosolic proteins remained constant under all conditions assayed. These data suggest that specific directions of stretch may play a crucial role in regulating MF organization and the metabolism of contractile proteins in the cardiac myocyte. The full text of this article is availabale at http://www.circresaha.org.


Key Words: stretch • myofibril • hypertrophy


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
The intact myocardium is composed of a complex array of muscle cells that are distributed in a series of discrete, overlapping cellular layers. Within each cell layer, the rod-like myocytes of the heart are distributed in parallel with one another along a common axis. In the subendocardium and subepicardium of the left ventricle, the myocytes are arranged in a longitudinal orientation that is in parallel with the long axis of the heart.1 The muscle cells of the midwall radiate in a circumferential pattern around the ventricular lumen and are oriented {approx}90° off-axis with respect to the more superficial and deep cell layers.2 Adjacent cell layers are interconnected and tethered to one another by an elaborate network of collagen fibrils.3

As a result of the intricate organization of the intact heart, cardiac myocytes are subjected to a very complex set of mechanical forces during the contractile cycle. For example, in working hearts, the axis of shortening during contraction varies considerably less than the local myofiber direction.2 4 5 The data from these experiments indicate that any given local population of myocytes may be exposed to a unique combination of mechanical signals. The mechanical events associated with cardiac function and ventricular wall stretch have been implicated in regulating cardiac gene expression,6 7 protein metabolism,8 9 and myofibrillar organization.10 11 12

There is circumstantial evidence from in vivo observations to suggest that cardiac myocytes are sensitive to the identity of the mechanical insult that applies an episode of stretch across the ventricular wall. For example, a sustained increase in cardiac preload or afterload is associated with an increase in ventricular wall stretch. However, a selective increase in either cardiac preload or afterload promotes very different changes in ventricular and cellular architecture,13 protein metabolism,14 15 and the mechanical indicators of cardiac performance.16 17 18 During the progression of eccentric and concentric hypertrophy, there are also regional changes in cardiac myocyte cell size19 20 21 22 and isoenzyme metabolism.23

In the present study, we have adapted the aligned myocyte cell culture system24 to examine how cardiac myocytes respond to specific directions of stretch in vitro. Neonatal cardiac myocytes maintained in this culture system typically exhibit an in vivo, rod-like cell shape with myofibrils that are distributed in parallel with the long axis of the cell. As a population, these rod-like cells are aligned in parallel with one another in a tissue-like pattern of organization. These characteristics make it possible to selectively apply different degrees and directions of stretch across the myofibrils. Applying a sustained static stretch of up to 10% in parallel with the long axis of aligned myocytes did not alter myofibrillar alignment, the turnover of myosin heavy chain (MHC) or actin, or the total cellular concentration of these contractile proteins. In contrast, even a modest degree of stretch across the short axis of the cells initiated changes in myofibrillar alignment, suppression in the turnover of both MHC and actin, and an increase in the total cellular concentration of these two contractile proteins. Cultures of stellate-shaped myocytes that displayed random arrays of myofibrils had a response that was intermediate to the cultures of aligned myocytes. Cardiac myocyte response to stretch was independent to the contractile state of the cells. These data suggest that specific directions of stretch may regulate the accumulation and posttranslational metabolism of contractile proteins in the cardiac myocyte.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Cell Isolation
Reagents were purchased from Sigma Chemical Co unless otherwise noted. Cardiac myocytes were enzymatically dissociated from 3- to 4-day-old neonatal rats according to the methods of Simpson et al.12 Cells were suspended and plated into culture in DMEM (Gibco) supplemented with 8.0% horse serum (Flow Laboratories), 5.0% newborn BSA (Gibco), and cytosine arabinoside (10 µg/mL).

Substrate Preparation
Silastic rubber substrates (Specialty Manufacturing) were prepared for cell culture according to the methods of Simpson et al.12 Static stretching devices (Figure 1ADown) were immersed in double-distilled H2O and autoclaved. Sterile silastic rubber membranes were mounted into static stretching devices and placed into sterile 100-mm culture dishes. Thin gels of aligned type I collagen were prepared according to the methods of Simpson et al.24 In brief, 500 µL of 10x MEM (Gibco) was mixed with 500 µL 200 mmol/L HEPES and placed on ice, final pH 7.0 to 7.4. A 3.5-mL layer of collagen type I (3.0 mg/mL; Celtrix) was applied over the top of this solution and mixed by inversion. This solution was then diluted to 10 mL with ice-cold, serum-free MEM (final concentration of collagen 1.05 mg/mL). This formulation is subsequently referred to as stock collagen solution. Thin gels of aligned collagen were prepared by applying 175 µL stock collagen solution to the edge of a silastic membrane. The collagen was then drawn across the substrate with a sterile cell scraper using a single, continuous stroke. The stretching devices were tipped at a low angle (<5°) to facilitate the flow of collagen across the silastic membrane during this procedure. Subsequently, the devices were tipped at a 45° angle, and the collagen was allowed to drain across the membrane along the axis in which it was originally applied. Excess collagen was aspirated, and the 100-mm culture dishes containing the static stretching devices were transferred to a 37°C incubator for 60 minutes. At the conclusion of the incubation period, the dishes were removed from the incubator, and the collagen was allowed to dry down onto the silastic membranes in a sterile laminar flow hood. These procedures resulted in the formation of a thin film of collagen consisting of fibrils that were preferentially oriented along the axis of application (Figure 1BDown). Nonaligned gels of collagen were produced by pipetting stock collagen onto the rubber membranes and swirling the dish several times. The excess collagen was aspirated. This procedure produced a tangled network of collagen fibrils that lacked a clearly defined orientation (Figure 1CDown).



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  		Figure 1. A, Static stretching device. Static stretching devices were constructed of nylon and consisted of a small frame with two axles separated by lateral supports. The lateral supports were threaded to accept calibrated thumb screws. Silastic membranes were mounted onto the devices by two friction-fit C-clamps. Bar = 40 mm (panel A only). B, Thin gel of aligned collagen. Thin gels of aligned collagen consisted of a partial film of fibrils. Double-headed arrow denotes the axis of application. C, Thin gel of random collagen. Random thin gels of collagen were composed of a tangled network of fibrils with no discernible polarity.

Cell Culture
Myocytes were suspended at a concentration of 2.0x106 cells/mL and plated onto a silastic membrane mounted in a static stretching device (1.75 to 2.0x106 myocytes/cassette). A small retaining ring was used to facilitate cell plating; rings were removed before experimentation.12 The cells were allowed to adhere for 24 to 36 hours; the retaining rings were then removed and the cultures were rinsed with serum-free DMEM and refed serum-supplemented culture medium. Cultures were subsequently fed at daily intervals. Experimentation was initiated on day 4 of culture and completed 24 hours later.

Protein Turnover Studies
On day 4 in vitro (ie, after 96 hours of maintenance culture), cultures of spontaneously beating myocytes were rinsed in serum-free, methionine-free DMEM and biosynthetically labeled for 2 hours with trans-labeled [35S]methionine (5 µCi/mL; ICN Biomedicals) prepared in methionine-deficient, serum-supplemented DMEM culture medium (pulse-labeling interval). At the conclusion of the pulse-labeling period, the cells were rinsed 3 times in a large volume of serum-free DMEM (30 mL) and transferred to serum-defined chase medium (1:1:1, DMEM:F12:PC-1; supplemented with 2 mmol/L unlabeled methionine, 3 mmol/L glutamine, 100 U/mL Fungizone, 100 U/mL streptomycin, 100 U/mL penicillin, and 10 µg/mL cytosine arabinoside). PC-1 medium was purchased from Hycor Biomedical Corporation. This formulation was selected to match as closely as possible the conditions that we have previously used to study the turnover of MHC25 and actin26 in cultured neonatal heart cells subjected to different loading conditions.12

At the onset of the chase interval, biosynthetically labeled cells were subjected to a 0%, 0.5%, 1.0%, 2.5%, 5.0%, or 10% sustained static stretch and cultured for an additional 24 hours. At the conclusion of a 24-hour chase period, the different treatment groups were rinsed 3 times in ice-cold, serum-free DMEM supplemented with 2 mmol/L unlabeled methionine. The myocytes were then extracted and scraped into 30 to 35 mL of low-salt buffer (LSB [in mmol/L]: NaCl 40, Na2PO4 5.0, MgCl2 1.0, DTT 1.0, EGTA 0.1, PMSF 1.0, DTT 1.0, and 0.1% Triton X-100), quantitatively transferred to a 50-mL centrifuge tube, and centrifuged (15 minutesx12 000g, 4°C). Soluble and insoluble fractions were separated. The cell pellets were resuspended in 50 µL Nanopure-filtered H2O (Barnstead), diluted to a final volume of 200 µL in sample buffer (62.5 mmol/L Tris-HCl, pH 6.8, plus 5.0% ß-mercaptoethanol, 10.0% glycerol, and 8.0% SDS), and boiled for 10 minutes.

A Packard 1500 Tri Carb liquid scintillation counter (Packard Instruments) was used to determine the relative amount of radioactivity present in the soluble and insoluble protein fractions. Within each experiment, the amount of protein-bound radioactivity present in stretched cells was expressed as the percentage of the radioactivity remaining in unstretched, beating controls.12 The means of different degrees of stretch were compared by Fisher’s protected test for the least-significant difference when one-way ANOVA indicated that a given direction of stretch had an effect at P<=0.05. Similar methods were used to assess the turnover and accumulation of specific proteins in response to stretch.

The relative concentrations of MHC and actin were quantitatively analyzed by SDS-PAGE and laser densitometry. Protein bands corresponding to MHC (206 kDa) and actin (43 kDa) were identified by calculating the relative mobility of candidate proteins on SDS polyacrylamide gels in relation to molecular mass markers. In each experiment, an equal volume of the low-salt insoluble fraction was separated on a single 130-mm-long, 1.5-mm-thick, 10.0% SDS polyacrylamide slab gel. The gels were fixed and stained overnight with Coomassie Brilliant Blue (Figure 2Down). Destained gels were scanned with an LKB Ultrascan XL laser densitometer (Bromma, Sweden). Each protein band of interest was scanned 3 times, and the average area under each band was calculated by autointegration (Gelscan XL software, LKB). The relative amount of MHC and actin present in the different treatment groups was expressed in arbitrary units of optical density as a percentage of MHC or actin remaining in unstretched, beating controls.



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  		Figure 2. Representative SDS gels and autoradiograms. SDS gels (left-hand column) of the LSB-insoluble protein fraction of cultured myocytes maintained under different loading conditions. Equal volumes of extract were loaded into each lane. Random cells (A), aligned cells stretched in parallel with the myofibrils (B), and aligned myocytes stretched across the short axis of the myofibrils (C). Representative autoradiograms for each SDS gel are depicted in the right-hand column for each treatment condition. Note that exposure in the linear range could not always be achieved for MHC and actin on the same exposure. In experiments during which this occurred, repeated autoradiographic exposures were taken to independently tailor film exposure to the linear range for MHC (206 kDa) and actin (43 kDa). Degree of stretch placed across the cultures is indicated in panel C.

The relative amount of biosynthetically labeled MHC and actin was determined by separating equal volumes of insoluble cell extract on a single 10.0% slab gel as described. The gels were then processed for autoradiography with fluorographic enhancement (Figure 2Up). Autoradiograms were exposed for varying lengths of time to obtain data for MHC and actin in the linear range of autoradiographic exposure. The relative amount of biosynthetically labeled MHC and actin present in the different treatment groups was expressed in arbitrary units of optical density as a percentage of biosynthetically labeled MHC or actin remaining in unstretched, beating controls.

Pharmacological Experiments
To examine the effects of contractile activity on the response of cardiac myocytes to different directions of stretch, we conducted pulse-chase experiments in the presence and absence of isoproterenol (ISO; 1x10-6 mol/L) or nifedipine (NIFED; 15x10-6 mol/L). Replicate cultures of myocytes were pulse-labeled for 2 hours with [35S]methionine. The cultures were rinsed 3 times in serum-free DMEM and stretched 2.5% either in parallel with the long axis (longitudinal stretch) or across the short axis (perpendicular stretch) of myofibrils. Myocyte cultures were then transferred to control, serum-defined PC-1 chase medium or chase serum–defined PC-1 chase medium supplemented with NIFED or ISO for 24 hours. At the conclusion of the chase interval, cultures were processed as described in the protein turnover studies. The degree of stretch used in these experiments was determined empirically from the experiments summarized in Figures 6Down and 7Down; this degree of stretch across the short axis of aligned myocytes had the maximal impact on protein metabolism. The application of 15x10-6 mol/L NIFED to cultured myocytes is sufficient to initiate immediate and sustained contractile arrest in the cells.12 ISO at 1x10-6 mol/L induces accelerated beating and, with time, hypertrophy in cultured neonatal26 and adult cardiac myocytes.27 Data were normalized to the relative amount of protein-bound radioactivity observed in unstretched cultures of spontaneously beating myocytes and were pooled from 4 experiments. Two-way ANOVA was used to examine the effects of NIFED or ISO treatment in the presence of specific directions of stretch. A Tukey test for a pairwise multiple comparison was used to test for differences among the different treatment groups.



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  		Figure 6. Protein metabolism in aligned myocytes stretched in parallel with the myofibrils. The total amount of protein-bound radioactivity present within the myofibrillar protein fraction and the cytosolic fraction was not altered by stretch in parallel with the myofibrils. The concentrations of biosynthetically labeled MHC and actin also remained constant. A modest increase in the total cellular concentration of MHC and actin in cells stretched 2.5% with respect to cells stretched 1.0% was observed. Bars indicate P<0.05 differences among samples. Dotted line indicates baseline controls. See also Figure 2Up for representative raw data.



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  		Figure 7. Protein metabolism in aligned myocytes stretched across the short axis of the myofibrils. A static load of >1% and <10% suppressed the loss of radioactivity from the myofibrillar protein fraction. Intermediate degrees of stretch also suppressed the loss of biosynthetically labeled MHC and actin from the cultures. These metabolic changes were associated with the accumulation of these contractile proteins. The metabolism of the LSB fraction remained constant under all loading conditions. Bars indicate P<0.05 differences among samples. Dotted line indicates baseline controls.

Confocal Microscopy
Myocytes were cultured under the different loading conditions described in the previous sections. All samples were processed in situ on silastic membranes installed in the static stretching devices. Cultures were rinsed in Moscona’s saline supplemented with 50 mmol/L KCl, fixed for 10 minutes in 2.0% paraformaldehyde prepared in Sorensen’s PBS, and extracted for 10 minutes in 0.5% Triton X-100 prepared in PBS. Extracted cells were washed in PBS and stained with diluted rhodamine phalloidin (1:100; Molecular Probes). Cultures were mounted in PBS-glycerol plus DABCO (3:1 PBS-glycerol, 1 mg/mL DABCO). Laser scanning confocal microscopic analysis was performed with a BioRad MRC 1000 (BioRad Microsciences).


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Myofibrillar Structure and Protein Metabolism in Random Cells Subjected to Various Degrees of a Sustained, Static Stretch
Myocytes plated onto a random matrix of collagen spread out during the first 24 to 48 hours of culture, expressed a stellate-shaped cell, assembled myofibrils, and began to beat spontaneously. After 5 days of culture, stellate myocytes stained with rhodamine phalloidin displayed well-differentiated myofibrils (Figure 3ADown and 3BDown). The contractile filaments were in close apposition with one another and radiated throughout the sarcoplasm. Within any given subcellular domain, the myofibrils exhibited a high degree of lateral registry. However, the myofibrils of adjacent domains were frequently arrayed along a completely different axis with respect to the myofibrils present in the first domain.



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  		Figure 3. Myofibrillar organization in stellate cardiac myocytes subjected to different loading conditions. After 96 hours of culture, spontaneously beating myocytes exhibited arrays of densely packed myofibrils (A and B). A, Low-magnification image of a field of random myocytes. B, High-magnification image of myofibrillar organization in the spontaneously beating cell. Applying a 1% static stretch for 24 hours did not promote any dramatic changes in myofibrillar organization or sarcomere structure (C). At a static load of 5%, the lateral alignment of the myofibrils deteriorated, and the contractile filaments began to exhibit a branching pattern (D). As the static load was increased to 10%, the myofibrils displayed an increasingly complex branching pattern, the lateral alignment of adjacent myofibrils was lost, and the percentage of cells that exhibited focal damage to the myofibrils increased (E and F). Myocytes stretched 10% often displayed myofibrils with an "undulating" or wavy pattern (E). Panel F illustrates the region indicated by the asterisk in panel E at higher magnification. This image depicts focal damage along a series of myofibrils; these regions stain continuously with rhodamine phalloidin and lack the banded pattern of mature sarcomeres (arrows). This type of damage did not appear to be confined to any particular domain or plane of focus within the myocytes. Arrowheads in panel F identify a branch point along a myofibril. Bar=100 µm (A), 8.5 µm (B through E), and 10 µm (F). Double-headed arrow in panel E indicates direction of stretch for all panels.

Applying a modest degree of stretch to stellate myocytes did not initiate any dramatic changes in the overall organization or structure of the myofibrils (Figure 3CUp). The contractile filaments of cells stretched 1.0% or less remained dispersed throughout the sarcoplasm and continued to display a random orientation with respect to another. At a static load of 5% or greater, the lateral alignment of the myofibrils deteriorated, and many filaments displayed a branching pattern (Figure 3DUp). Regions along some of these filaments stained continuously with rhodamine phalloidin. The majority of stretched cells accumulated a population of smaller-diameter phalloidin-positive filaments that stained in a periodic pattern. In cells stretched 10%, the myofibrils exhibited a complex branching pattern, and the relative percentage of cells with contractile filaments with domains that stained continuously with rhodamine phalloidin increased (Figure 3EUp and 3FUp). These nonstriated regions appeared to be continuous with adjacent myofibrils and were often interconnected on either end with contractile filaments that exhibited the banded staining pattern that is typical of mature sarcomeres.

In pulse-chase experiments, the posttranslational metabolism of random myocytes displayed a threshold response to an external load (Figure 4Down). Myocytes stretched 1.0% to 5.0% retained on average 50% to 100% more radioactivity in the total myofibrillar protein fraction (LSB-insoluble proteins) at the end of the experimental interval than spontaneously beating, unstretched controls. The amount of radioactivity present in the myofibrillar protein fraction in myocytes stretched 0.5% or 10% was not statistically different from controls (0.0% stretch). In general, the posttranslational metabolism of MHC and actin was regulated in parallel with the metabolism of the total myofibrillar protein pool. The data indicate that a 2.5% stretch was most effective at suppressing the loss of biosynthetically labeled MHC and actin from the myocytes. This magnitude of stretch also increased the total cellular concentration of these contractile proteins. In contrast to these results, the total amount of protein-bound radioactivity present in association with proteins isolated from the cytoplasmic fraction (LSB-soluble proteins) was not affected by the application of an external load.



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  		Figure 4. Protein metabolism in stellate cardiac myocytes subjected to different loading conditions. Cells stretched 2.5% retained more radioactivity in the myofibrillar protein fraction than unstretched controls and cells stretched 0.5% or 10%. This degree of stretch was also most effective at suppressing the loss of biosynthetically labeled MHC and actin from the cultures. A static load of 2.5% also increased the total cellular concentration of MHC and actin with respect to controls. The metabolism of the cytosolic fraction remained constant in all treatment groups. Data are normalized and expressed as a percentage of protein-bound radioactivity or optical density observed in unstretched controls. Bars in each graph indicate differences that are significant at P<=0.05 at a static load of 2.5% with respect to other loading conditions. Dotted line indicates baseline controls. See Figure 2Up for representative SDS gels and autoradiograms of this summarized data.

Myofibrillar Structure and Protein Metabolism in Aligned Myocytes Subjected to a Sustained, Static Stretch in Parallel With the Myofibrils
Myocytes plated onto a thin gel of aligned collagen spread on the underlying collagen fibrils and expressed an elongated, rod-like cell shape. Staining with rhodamine phalloidin revealed myofibrils densely packed in the sarcoplasm and arrayed in parallel with the long axis of the cells (Figure 5Down). As a population, the rod-like myocytes were distributed along a common axis. This tissue-like pattern of organization made it possible to assay how specific directions of stretch affected cardiac myofibrillar structure and protein metabolism.



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  		Figure 5. Myofibrillar organization in aligned myocytes subjected to different degrees of stretch. Control, unstretched cells (A, low magnification; B, high magnification). Aligned myocytes exhibit a rod-like cell shape and polarized arrays of myofibrils. Cells stretched in parallel with the myofibrils (C, E, and G) and across the short axis of these filaments (D, F, and H). Cells in panels C and D=2.5% stretch; cells in panels E and F=5.0% stretch; cells in panels G and H=10% stretch. The myocytes of cultures subjected to modest degrees of stretch in parallel with their long axis retained linear arrays of myofibrils (C=2.5% stretch, E=5.0% stretch). As the magnitude of stretch increased (G=10% stretch), the myofibrils accumulated regions that stained continuously with rhodamine phalloidin (G, arrows), but the contractile filaments retained their polarity. Stretch across the short axis of aligned myocytes was associated with the accumulation of branching myofibrils and domains that stained continuously with rhodamine phalloidin (arrowheads, D). As the degree of stretch was increased, the lateral registry of the myofibrils showed evidence of deterioration (F and H). At 10% strain (H), branching myofibrils were prominent, and the myofibrillar alignment was lost. Bar=100 µm (A), 10 µm (B), and 10 µm (C through H). Arrow in panel A indicates direction that collagen was applied to the silastic membrane to produce the control cultures. Double-headed arrow in panel G denotes direction of stretch applied across the cultures depicted in panels C through H.

Applying a sustained, static stretch in parallel with the long axis of the myofibrils in aligned myocytes did not alter the alignment or lateral registry of these filaments. The myofibrils in cells stretched <5% and stained with rhodamine phalloidin displayed the banded pattern that is typical of mature sarcomeres in the cultured myocyte. In cells stretched >=5%, we observed evidence of structural abnormalities in the myofibrils (Figure 5Up). At irregular intervals along some myofibrils, there were domains where sarcomeres appeared to be missing. Regions along the myofibrils that stained continuously with phalloidin also were encountered.

In contrast to random cells, the application of a sustained, static stretch in parallel with the myofibrils of aligned myocytes was not associated with any change in the posttranslational metabolism of contractile proteins (Figure 6Up). Sustained static loads of <=10% did not alter the amount of biosynthetically labeled protein present in the total myofibrillar protein fraction. The posttranslational metabolism of MHC and actin also remained constant at all levels of static stretch. There was a modest increase in the total cellular concentration of MHC and actin in cells stretched 2.5% with respect to cells stretched 1.0%. This difference appears to arise from a combination of factors. Over several experiments, cells stretched in parallel with the myofibrils exhibited a very uniform response to this type of strain. At a static load of 2.5%, the cells exhibited a modest increase in the concentration of MHC and actin whereas a 1.0% static stretch appeared to decrease the concentration of these proteins. The total amount of protein-bound radioactivity present in the cytosolic fraction of the cultures was not altered by stretch in parallel with the myofibrils.

Myofibrillar Structure and Protein Metabolism in Aligned Myocytes Subjected to a Sustained, Static Stretch Across the Short Axis of the Myofibrils
Aligned myocytes subjected to moderate degrees (<5%) of static stretch across the short axis of the myofibrils did not display any dramatic changes in myofibrillar organization or in the architecture of individual sarcomeres (Figure 5Up). Regions that stained continuously with rhodamine phalloidin were encountered; however, these structures did not appear as frequently in these cells as they did in myocytes that had been stretched in parallel with the myofibrils. As the degree of stretch was increased >5%, an increasing number of myofibrils began to display regions that stained continuously with rhodamine phalloidin. At the highest levels of static stretch that we assayed (10%), the lateral borders of the rod-like myocytes appeared to be distorted, and the lateral registry of the myofibrils underwent deterioration. In extreme examples, the contractile filaments and sarcomeres were in disarray.

Stretch across the short axis of aligned myocytes suppressed the posttranslational metabolism of proteins present in the myofibrillar protein fraction in a pattern that was qualitatively similar to that observed in the random cultures (compare Figures 4Up and 7Up). However, these cells appeared to be more sensitive to stretch than random cells; a sustained, static stretch of >0.5% and <10% suppressed the loss of biosynthetically labeled proteins from this protein fraction with respect to controls and cells stretched 0.5% (Figure 7Up). The processes that regulated the turnover and accumulation of MHC and actin were regulated in parallel with the total myofibrillar protein fraction. In contrast to these results, once again, the total amount of protein-bound radioactivity present in the cytosolic fraction of the cells remained constant under all degrees of static stretch that we examined.

Interactions Between Contractile Activity and Specific Directions of Stretch
To test for potential interactions between specific directions of stretch and the spontaneous contractile activity of cultured myocytes, we used ISO (1x10-6 mol/L) and NIFED (15x10-6 mol/L) to manipulate the frequency of contraction in cultures of aligned myocytes. Increasing the rate of contraction with the addition of ISO did not alter total myofibrillar protein turnover in control, unstretched myocytes (Figure 8ADown). Stretching aligned myocytes in parallel with the myofibrils in concert with ISO treatment accelerated total myofibrillar protein turnover {approx}25%. In contrast, when aligned cultures were stretched across the short axis of the myofibrils and treated with ISO, total protein turnover in the total myofibrillar protein fraction was suppressed up to 75%. The total amount of protein-bound radioactivity in the LSB fraction of the cultures remained constant under all of the conditions that were assayed (Figure 8BDown).



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  		Figure 8. Interactions between the contractile state of cardiac myocytes and specific directions of stretch in aligned cultures. A, Summary of the posttranslational metabolism of the myofibrillar protein fraction in these cultures. The regulation of protein turnover was most closely associated with the direction of stretch placed across the myofibrils. No statistically significant changes in protein turnover were associated with ISO treatment in unstretched cultures (ISO). Myofibrillar protein turnover was modestly accelerated in myocytes treated with ISO and subjected to strain in parallel with the myofibrils (L+ISO). In contrast, myofibrillar protein turnover was suppressed in cultures treated with ISO and stretched across the short axis of these filaments (P+ISO). Contractile arrest accelerated protein turnover in unstretched myocytes (NB). Protein turnover remained accelerated in arrested cultures subjected to stretch in parallel with the myofibrils (L+NB). Stretch across the short axis of the myofibrils suppressed the accelerated turnover of myofibrillar proteins normally observed when cultured myocytes are subjected to contractile arrest (P+NB). B, Turnover of proteins in the nonmyofibrillar protein fraction. Turnover in this protein pool remained constant under all loading conditions, regardless of the contractile state of the cells or the direction of stretch imposed on the cultures. Dotted line indicates baseline controls. *P<0.05 for cells treated with ISO with respect to controls. **P<0.05 for cells treated with NIFED with respect to controls.

In previous studies, we have demonstrated that contractile arrest with the L-type calcium channel blocker NIFED accelerates the turnover of myofibrillar proteins in random cultures of cardiac myocytes.12 Stretch was found to suppress the accelerated turnover of contractile proteins induced by contractile arrest. Treatment of aligned cardiac myocytes with 15x10-6 mol/L NIFED resulted in immediate and sustained contractile arrest. Total myofibrillar protein turnover was accelerated {approx}30% in nonbeating, unstretched cultures with respect to control, spontaneously contracting cultures (Figure 8AUp). Aligned cultures stretched in parallel with the myofibrils in tandem with NIFED treatment exhibited a similar acceleration in total myofibrillar protein turnover (Figure 8AUp). In contrast to these results, stretch across the short axis of the myofibrils in NIFED-treated cultures substantially overcame the effects of contractile arrest. Total myofibrillar protein turnover in control, unstretched, spontaneously beating cultures and nonbeating cultures stretched across the short axis of the myofibrils was identical. As before, the turnover of proteins in the LSB fraction of the cells remained constant in the different treatment groups, regardless of the direction of stretch that was applied or the contractile state of the cells (Figure 8BUp).


*    Discussion
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 *Discussion
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The metabolism of a cardiac myocyte is dynamically coupled to the mechanical forces experienced by the cell.28 Any sustained increase or decrease in mechanical activity provokes a highly integrated response that ultimately leads to the hypertrophy or atrophy of the cell. This adaptive program allows the myocyte to balance its mass and performance characteristics with the prevailing workload. Cardiac myocytes are routinely subjected to a complex mixture of biomechanical signals; however, ventricular wall stretch appears to be the mechanical factor most closely linked to the overall regulation of protein metabolism in the heart.8 9 It can be inferred from our in vitro data and in vivo observations that cardiac protein metabolism is regulated by the magnitude as well as the temporal and spatial context in which stretch is applied across the cell.

It is clear that the actual extent to which a cardiac myocyte will undergo hypertrophy is not solely dictated by the magnitude of a mechanical insult. The temporal context, or the interval of time that changes in ventricular wall stretch occur during the contractile cycle (ie, diastole or systole), appears to play a role in shaping how ventricular myocytes respond to a mechanical signal. A selective and sustained elevation in preload initiates changes in ventricular wall architecture that are separate and unique from those that evolve in response to a sustained increase in cardiac afterload, even in hearts that have been subjected to comparable amounts of stroke work.29 In the present study, we have held the temporal context in which we apply stretch constant by using a sustained, static load. Our data indicate that specific directions and degrees of stretch regulate myofibrillar protein accumulation, contractile protein turnover, and sarcomere structure in the cultured myocyte. We were unable to alter the response of the cells to stretch by manipulating the contractile state of the cells (Figure 8Up). Cultured myocytes responded in a very characteristic fashion to stretch, regardless of the contractile state of the cells (ISO or NIFED treated). These results indicate that the detection/transduction system that enables cultured myocytes to discriminate between different directions of stretch operates independently of the length-tension relationship that ordinarily governs the mechanical performance and growth of striated muscle.

There is evidence that cardiac myocytes can accumulate myofibrillar proteins through a posttranslation mechanism that does not appear to be directly regulated by an intracellular signal cascade in the conventional sense. The data indicate that cardiac myofibrils can physically sequester contractile proteins from targeting for proteolysis.25 26 30 31 32 Interventions that disrupt the structure of these filaments accelerate the turnover of MHC and actin.12 25 26 Even in artificial systems, isolated myofibrils appear to segregate contractile proteins from proteolysis.33 The structure of the cardiac myofibril is very sensitive to changes in contractile activity and mechanical tension.10 11 12 26 31 It is entirely possible that specific directions of stretch can indirectly regulate the accumulation and posttranslational metabolism of contractile proteins by directly regulating the rate at which these structural proteins are assembled into a myofibril. The small-diameter branching myofibrils that accumulate in myocytes undergoing hypertrophy in response to stretch (Figures 4DUp and 5DUp, 5F, and 5H) may represent nascent myofibrils in the early stages of assembly.11 26

Stretch also has an impact on the organization of preexisting myofibrils and can stabilize the structure of these filaments, even in the absence of spontaneous contractile activity in the cultured cardiac myocyte.12 In the stellate myocyte, maximal growth was elicited at moderate levels of stretch. At higher levels of stretch (10%), some myofibrils appeared to be damaged, protein turnover remained constant, and the concentration of total cellular MHC remained unchanged. This biphasic response may indicate that stretch has multiple effects. We would predict from our results with aligned myocytes that individual myofibrils of stellate myocytes that experience a cross-fiber strain would be stabilized. Filaments arrayed in parallel with the strain may be subjected to eccentric loading and damage. In these cells, the hypertrophic and potentially damaging effects of stretch may compete with one another. In skeletal muscle, severe eccentric loading (stretch in parallel with myofibrils) promotes the evolution of sarcomeric anomalies34 and initiates an acceleration in contractile protein turnover.35 This interpretation is consistent with our observations that high levels of strain placed in parallel with the myofibrils of aligned myocytes initiate sarcomere anomalies. Furthermore, increasing the mechanical load on these cultures with the addition of ISO markedly accelerated the loss of radioactive tracer from the aligned cells (Figure 8Up).

We have assumed in our analysis that our pulse-chase experiments faithfully report the posttranslational metabolism of cardiac proteins. However, a technical consideration that must be addressed in any pulse-chase experiment is the assumption that no treatment causes a preferential loss of cells from the cultures. Cell loss artificially depletes cultures of protein-bound radioactivity and inflates the apparent rate of protein turnover. We believe the metabolic profile of the cytosolic fraction (LSB soluble) argues against this type of artifact. The turnover of this protein pool remained constant under all conditions (Figures 4Up, 6Up, and 8Up). These data do not indicate a lack of regulation; rather, these results suggest that contractile function and mechanical activity do not regulate the rate-limiting step in the turnover of this heterogeneous protein pool. If the turnover of this protein pool is unaffected by contraction or stretch, the selective loss of cells from some cultures would be reflected in a reduction in the amount of radioactivity present in the cytosolic pool of the affected cultures. Because the amount of radioactivity in the cytosolic pool remained constant, we conclude that our data faithfully report the relative amount of contractile protein turnover occurring in our cultures.

To our knowledge, no concerted effort has been made to examine the relationships that might exist between the local myofiber direction within the intact heart and the response of individual myocytes to changes in ventricular wall stretch. We propose that a vectorial summation of the mechanical signals that a local population of myocytes receives during the cardiac cycle defines a primary axis of stretch for each region of the heart. We must emphasize that we do not believe that ventricular myocytes are exposed to a single, well-defined direction of stretch. Instead, the response of each cell is determined by the integration of many different mechanical, and biochemical, signals. Even in a single region of ventricular wall, the long axis of myocytes rotates going from the epicardial to endocardial surface. Any perturbation that alters cardiac preload or afterload must also alter the relative balance of these forces and reinforce, or disrupt, the axis of stretch that is placed across a local population of myocytes. In our model, the spatial context, or the direction that an episode of stretch is applied across the ventricular myocyte, interacts with the magnitude and temporal context (diastole versus systole) of the mechanical insult to regulate cardiac metabolism and growth.

The global changes in ventricular structure that occur in eccentric and concentric hypertrophy are accompanied by regional variations in the hypertrophic response.19 21 22 36 We believe that the spatial context of the mechanical signals that are applied to different areas of the heart may contribute to this regional variation and ultimately shape the architecture of the ventricular wall. For example, in a volume overload, the ventricle is deformed5 and dilates along the circumferential axis of the heart.37 With time, the ventricular wall becomes less stiff and is preferentially distended along this same axis during diastolic filling.38 The ventricular myocytes of the midwall are arrayed in parallel with the circumferential axis of the heart.1 The relative amount of stretch that these cells experience along an orientation that is nearly in parallel with the myofibrils must increase as the ventricle progressively dilates. During the early stages of an acute volume overload, there is no change in the synthesis of total protein14 or MHC.15 Our in vitro results would predict this type of response, because in our culture system, stretch in parallel with the myofibrils does not promote the accumulation of contractile proteins. However, applying a strain that gradually increases over time does promote myocytes to undergo cell elongation in vitro,39 a hallmark of eccentric hypertrophy in vivo.20 36 40

In contrast to cells of the midwall, ventricular myocytes of the endomyocardium are arrayed nearly in parallel with the long axis of the heart.1 2 We predict that the dilation of the ventricle, in concert with the changes in ventricular function that accompany eccentric hypertrophy, will chronically (during the dilation of the ventricle) and then systematically (each contractile cycle) increase the amount of stretch these cells experience in a cross-fiber direction. This is the direction of strain that promoted the maximal growth response in our culture system. In vivo, myocytes of the endomyocardium undergo a greater increase in cell size than myocytes residing in the midwall or epimyocardium during eccentric hypertrophy.19 21 41

In concentric hypertrophy, there is a selective increase in cell diameter, very little or no dilation of the ventricular lumen, and a marked increase in the diameter of the ventricular wall.42 Mapping regional stresses during the contractile cycle indicates that the principal axis of shortening varies considerably less than the local myofiber orientation.4 43 44 These data imply that an increase in afterload will increase the relative amount of cross-fiber strain that ventricular myocytes experience as they contract more forcefully to overcome the elevated workload. Thus, in our model, we argue that an increase in cross-fiber strain during systolic loading is a critical signal for promoting cardiac cell hypertrophy during the evolution of concentric hypertrophy. In our experiments, stretch across the short axis of aligned myocytes was most effective at promoting the accumulation of contractile proteins and branching myofibrils, phenotypic characteristics that are typically used to describe the cardiac myocytes of concentric hypertrophy.

There also is clear evidence of a temporal component to this response. Unlike an acute increase in cardiac preload, an acute increase in cardiac afterload is associated with an immediate acceleration in the synthesis of total protein14 and MHC.15 In vitro, an acute increase in afterload is much more effective than an acute increase in preload at accelerating protein synthesis in the isolated ventricular papillary muscle.45 Regional variations in the extent of the hypertrophic response also accompany concentric hypertrophy. The myocytes of the endomyocardium undergo a greater increase in cell size19 and express more ß-MHC than cells residing in the outer portion of the epimyocardium.22 These regional changes are clearly correlated with the magnitude of the wall stress observed along the luminal surface of the overloaded ventricle.29 46 However, the myocytes of the endomyocardium may be stimulated by a potent combination of mechanical signals as concentric hypertrophy develops: an increase in wall stress (magnitude), an increase in systolic loading (temporal), and stretch in the cross-fiber direction (spatial).

These observations, and our data, imply that cardiac gene expression and protein synthesis also are subject to regulation by specific directions of stretch. We believe that this information may be detected as a physical perturbation to a structural protein and transduced into an intracellular signal(s) that regulates myofibrillar protein metabolism. Any number of structural features may physically represent this putative detection/transduction system. However, it seems likely that any receptor for this type of information must have a unique and polarized distribution to allow for the detection of a specific direction of stretch. Matrix receptors of the integrin family and cell-cell adhesion molecules of the cadherin family fit this criterion. In the neonatal cardiac myocyte, the {alpha}1ß1 integrin is preferentially distributed along the peripheral domains of the Z-disks.47 The continuum that can be traced from the myofibrils through the cytoskeleton, integrins of the {alpha}1ß1 family, and constituents of the extracellular matrix physically propagates contractile forces across the sarcolemma.48 However, the concentration of {alpha}1ß1 integrins at the Z-disks may also allow them to serve as a discrimination and signal transduction system for the propagation of physical queues into the intracellular environment.49 This might be achieved by these receptors if one particular direction of stretch were more effective at deforming the membrane and bringing adjacent integrins into juxtaposition to initiate a signal cascade or directly propagate mechanical tension to the cytoskeleton. Isolated myocytes undergo a preferential increase in cell diameter during osmotic shock, suggesting that the plasma membrane may be constrained to some degree from undergoing deformation in parallel with the long axis of the rod-like myocyte.50

Myocytes plated onto thin gels of aligned collagen recapitulate many of the phenotypic characteristics that define the cytoarchitecture of the cardiac myocyte residing within the intact heart.24 51 52 Altering the integrin profile or disrupting the expression of cytoskeletal components that link these receptors to the myofibrils in the aligned myocyte leads to myofibrillar abnormalities, changes in cell shape, and perturbations in protein metabolism.51 52 The integrin profile of cardiac myocytes changes during normal development and with the progression of concentric hypertrophy.53 The characteristic regulation of integrin expression in the heart may represent a feedback loop in the signal cascade that ultimately regulates cardiac protein metabolism. Cell adhesion molecules represent another class of structural elements that are distributed in specific domains on the cardiac myocyte. The potential role of these molecules in the regulation of cardiac response to stretch is less clear. N-cadherin expression is regulated in a developmental pattern, and this molecule is critical for normal myofibril formation in the heart.54 Signal cascades initiated by the N-cadherin family of receptors can be modulated by integrin-mediated events,55 suggesting a possible mechanism for the integration of signals arising from different sources.

In summary, our data indicate that cardiac myocyte hypertrophy is stimulated in vitro by specific directions and degrees of stretch. At any given time, the rate of protein accumulation is determined by the rate at which a particular protein is synthesized and degraded. In our discussion, we have assumed that stretch concurrently suppresses protein turnover and accelerates protein synthesis. However, the relative contributions that protein synthesis and protein turnover play in regulating the cellular concentration of contractile proteins remains to be fully elucidated. If cardiac myocytes are sensitive to the spatial context that strain is placed across the ventricular wall, it could explain why partial ventriculectomy can be so effective at rescuing the dilated and failing heart.56 Surgically adjusting the geometry of the dilated heart clearly has an impact on the biomechanics of cardiac function, ie, improved pump function. However, in the long term, the removal of ventricular tissue also may alter the spatial pattern in which strain is applied across the ventricular wall. In turn, this may alter the primary axis of strain for myocytes of the midwall to an axis that is more conducive to cardiac cell growth and function. The myocytes of a failing heart that has been surgically altered appear to undergo hypertrophic growth.57


*    Acknowledgments
 
This work was supported in part by the National Institutes of Health (NIH) HL 58243 (D.G. Simpson), the American Heart Association, South Carolina Affiliate 9708274A (D.G. Simpson), and NIH HL 58893 (L. Terracio).

Received August 10, 1999; accepted October 7, 1999.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

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